Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase.

Hawrot, Edward; Kennedy, Eugene P.

doi:10.1073/pnas.72.3.1112

Cited by 90 publications

(60 citation statements)

References 18 publications

(11 reference statements)

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“…Previous results demonstrated that LacY either expressed in cells or reconstituted into proteoliposomes showed a near absolute dependence for uphill transport activity, but not downhill transport activity, on an ionizable amino-based zwitterionic phospholipid [PE or phosphatidylserine (PS)] (9,10,18,19). LacY expressed in cells in which the neutral glycolipid monoglucosyl diacylglycerol (GlcDAG) (20) but not diglucosyl diacylglycerol (GlcGlcDAG) (21) replaced PE also carried out uphill transport.…”

Section: Resultsmentioning

confidence: 99%

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Bogdanov¹,

Heacock²,

Guan

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

115

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Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

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Section: Resultsmentioning

confidence: 99%

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Bogdanov¹,

Heacock²,

Guan

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

115

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“…A temperature sensitive mutation in the psd locus was isolated by mutagenesis followed by brute force assay of individual colonies for PS decarboxylase (Psd) activity at an elevated temperature [63]. The initial mutant contained a second mutation, which rendered the cells temperature sensitive for growth but not for PS accumulation or lack of PE.…”

Section: Isolation Of Mutants In Phospholipid Metabolismmentioning

confidence: 99%

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

Dowhan¹

2013

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

108

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Although the study of individual phospholipids and their synthesis began in the 1920’s first in plants and then mammals, it was not until the early 1960’s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960’s. In 1970’s and 1980’s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990’s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems.

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“…In bacteria and yeast, PS is synthesized by a PS synthase (PSS) that utilizes CDP‐diacylglycerol (CDP‐DG) and l‐ serine (Ser) as substrates (CDP‐DG‐dependent PSS or CD‐PSS; EC 2.7.8.8; Matsumoto, 1997). In Escherichia coli , PS is rapidly converted to phosphatidylethanolamine (PE) by PS decarboxylase (PSD), so that little PS accumulates in cells (Hawrot and Kennedy, 1975). However, E. coli psd ts mutants accumulate high PS levels at non‐permissive temperatures, leading to cell lysis (Hawrot and Kennedy, 1978).…”

Section: Introductionmentioning

confidence: 99%

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

et al. 2011

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SUMMARYPhosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of L-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6% pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4¢,6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.

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Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase.

Cited by 90 publications

References 18 publications

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

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